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Monoclonal antibody of fluoroquinolone medicines as well as preparation method and application thereof

A monoclonal antibody and fluoroquinolone technology, applied in the field of immunochemistry, can solve the problems of expensive, unreachable, and unsuitable for universal application of instrument analysis methods, and achieve the effect of rapid and sensitive detection

Inactive Publication Date: 2012-05-23
深圳市华科瑞科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The current method for detecting fluoroquinolone drug residues in food is mainly based on instrumental analysis, but due to the high price of instrumental analysis and high requirements for operators, it is not suitable for popularization
However, the commonly used immunological detection method cannot achieve the purpose of detecting multiple drug residues through a single reaction because the antibodies obtained are often only directed against one or two fluoroquinolones.

Method used

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  • Monoclonal antibody of fluoroquinolone medicines as well as preparation method and application thereof
  • Monoclonal antibody of fluoroquinolone medicines as well as preparation method and application thereof
  • Monoclonal antibody of fluoroquinolone medicines as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Reagent and material preparation

[0023] Norfloxacin (National Institute for the Control of Pharmaceutical and Biological Products, 130450-200705), DMF (sigma, D4551), sodium acetate (sigma, S2889), acetic anhydride (Sinopharm Chemical Reagent Co., Ltd.), bovine serum albumin (BSA) (Jackson, 001-000-173), ovalbumin (OVA) (Sigma, A5503), Freund's complete adjuvant (Sigma, F5881), Freund's incomplete adjuvant (Sigma, F5506), tetramethylbenzidine (TMB) (Amresco, 0759), HAT (Sigma, H0262) and HT (Sigma, H0137), sodium chloride (Amresco, 0241), potassium chloride (Amresco, 0395), potassium dihydrogen phosphate (Sigma, P9791) , disodium hydrogen phosphate (Sigma, 71639), goat anti-mouse IgG-HRP (Jackson, 115-035-044), dimethyl sulfoxide (DMSO) (Applichem, 0231), polyethylene glycol 4000 (PEG4000) ( Sigma, P7306), DMEM high glucose medium (Gibco, 11995), fetal bovine serum (Gibco, C2027050).

[0024] Experimental animals and cells: Balb / c mice (6-8 weeks old, female) were p...

Embodiment 2

[0109] drug cross reaction test

[0110] The monoclonal antibody of norfloxacin and norfloxacin hapten structural analogue such as ciprofloxacin hydrochloride, ofloxacin, mesylate Flufloxacin, pefloxacin mesylate, difloxacin hydrochloride, enrofloxacin, lomefloxacin for competition experiments, dilute these standard products into different concentrations for indirect competition ELISA, draw inhibition curves, and calculate competitors IC 50 The results showed that the antibody had a high cross-reaction rate with the other five fluoroquinolones tested except for the low cross-reaction rate with dafloxacin and lomefloxacin , see Table 4 for the specific results.

[0111] type

[0112] Table 4

Embodiment 3

[0114] This example is an example of the application of the monoclonal antibody 4G11' in the establishment of an ELISA method for the detection of norfloxacin residues in the present invention, which can be used for the detection of fluoroquinolone residues in chicken, aquatic products, milk and honey. Taking chicken (purchased in the market) as an example, the main steps for detecting fluoroquinolone residues in chicken are as follows:

[0115] 1. Sample pretreatment: Weigh 15g chicken sample for homogenization, weigh 0.5g homogenate, add 1.5ml sample extract, shake and mix, centrifuge, take 100μL supernatant and 900μL sample diluent, mix, take 50μL for detection.

[0116] 2. The detection principle of the kit in the present invention is the indirect competition ELISA method. The original NOR-OVA is coated on a microwell plate, and 50 μL of 10, 5, 2.5, 1.25, 0.625 and 0.313 ng / ml Norfloxacin or the sample, then add 50 μL anti-fluoroquinolone monoclonal antibody 4G11', the re...

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Abstract

The invention relates to a monoclonal antibody as well as a preparation method and application thereof, in particular to a monoclonal antibody of fluoroquinolone drugs and application thereof, belonging to the technical field of immunochemistry. The monoclonal antibody of fluoroquinolones 4G11' is produced from a mouse hybridoma cell strain 4G11, and the subtype of 4G11 is an IgG1 type. The invention has the advantages that the monoclonal antibody 4G11' produced from the mouse hybridoma cell strain 4G11 has high cross reaction rate with various fluoroquinolone medicines and can be produced inlarge quantity and used for preparing an enzyme linked immunosorbent assay kit and colloidal gold test paper for detecting the fluoroquinolone medicines, achieves the purpose of quickly and sensitively detecting the residual fluoroquinolone medicines in milk, muscle and aquatic products and provides a basis for developing a kit for detecting various residual fluoroquinolone medicines.

Description

technical field [0001] The invention relates to a monoclonal antibody, its preparation method and application, especially the monoclonal antibody of fluoroquinolones, its preparation method and application, and belongs to the technical field of immunochemistry. Background technique [0002] Fluoroquinolones (FQs) are a new family of artificially synthesized bactericidal antibacterial drugs, which are widely used in animals because of their broad antibacterial spectrum, strong antibacterial activity, no cross-resistance with other antibacterial drugs, and low toxicity and side effects. and the treatment of various infectious diseases in humans. With the wide application of such drugs in food-borne animals, their residues have attracted widespread attention. In addition to its toxic and side effects, FQs residues in animal foods can easily induce drug resistance in human pathogenic bacteria, which further deepens people's understanding of the drug. In order to monitor the re...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/44G01N33/577
Inventor 唐宏王晓艳崔迎利王文夏文静倪同浩尹丽梅戴威韩晓娟林纪昀杨利郁靓汪纪宗
Owner 深圳市华科瑞科技有限公司
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